Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region.Abundant experimental evidence supports the commonsense notion that prolonged wakefulness decreases the degree of arousal, which is usually measured as electroencephalographic activation (EEG arousal to the duration of prior wakefulness (1). What might be the neural mediator of this effect of prior wakefulness? Our laboratory has provided evidence that the basal forebrain and mesopontine cholinergic neurons whose discharge activity plays an integral role in EEG arousal (2) are under the tonic inhibitory control of endogenous adenosine, an inhibition that is mediated postsynaptically by an inwardly rectifying potassium conductance and by an inhibition of the hyperpolarization-activated current (3). Adenosine is of particular interest as a putative sleep-wakefulness neuromodulator (4) because (i) the production and concentration of adenosine in the extracellular space have been linked to neuronal metabolic activity (5); (ii) neural metabolism is much greater during wakefulness (W) than during delta slow wave sleep (SWS) (6); and (iii) caffeine and theophylline are powerful blockers of electrophysiologically relevant adenosine receptors, promoting both subjectively and EEGdefined arousal while suppressing recovery sleep after deprivation (7). Our laboratory has recently demonstrated that microdialysis perfusion of adenosine in the cholinergic basal forebrain and the mesopontine cholinergic nuclei reduces wakefulness and EEG arousal (8).Although the preceding evidence is consistent with adenosine as a neural sleep factor mediating the somnogenic effects of prolonged EEG arousal and wakefulness, key questions relevant to a demonstration of this role have remained unaddressed. (i) Are brain extracellular adenosine concentrations higher in spontaneous W than in SWS? (ii) Do adenosine concentrations increase with increasing duration of W and then decline slowly as recovery sleep occurs after W? (iii) Do pharmacological manipulations increasing brain adenosine concentra...

Sleep is influenced by diverse factors such as circadian time, affective states, ambient temperature, pain, etc., but pathways mediating these influences are unknown. To identify pathways that may influence sleep, we examined afferents to the ventrolateral preoptic nucleus (VLPO), an area critically implicated in promoting sleep. Injections of the retrograde tracer cholera toxin B subunit (CTB) into the VLPO produced modest numbers of CTB-labeled monoaminergic neurons in the tuberomammillary nucleus, raphe nuclei, and ventrolateral medulla, as well as a few neurons in the locus coeruleus. Immunohistochemistry for monoaminergic markers showed dense innervation of the VLPO by histaminergic, noradrenergic, and serotonergic fibers. Along with previous findings, these results suggest that the VLPO and monoaminergic nuclei may be reciprocally connected. Retrograde and anterograde tracing showed moderate or heavy inputs to the VLPO from hypothalamic regions including the median preoptic nucleus, lateral hypothalamic area, and dorsomedial hypothalamic nucleus (DMH), autonomic regions including the infralimbic cortex and parabrachial nucleus, and limbic regions including the lateral septal nucleus and ventral subiculum. Light to moderate inputs arose from orexin and melanin concentrating hormone neurons, but cholinergic or dopaminergic inputs were extremely sparse. Suprachiasmatic nucleus (SCN) projections to the VLPO were sparse, but the heavy input to the VLPO from the DMH, which receives direct and indirect SCN inputs, could provide an alternate pathway regulating the circadian timing of sleep. These robust pathways suggest candidate mechanisms by which sleep may be influenced by brain systems regulating arousal, autonomic, limbic, and circadian functions.

We found previously that damage to a cluster of sleep-active neurons (Fos-positive during sleep) in the ventrolateral preoptic nucleus (VLPO) decreases non-rapid eye movement (NREM) sleep in rats, whereas injury to the sleep-active cells extending dorsally and medially from the VLPO cluster (the extended VLPO) diminishes REM sleep. These results led us to examine whether neurons in the extended VLPO are activated during REM sleep and the connectivity of these neurons with pontine sites implicated in producing REM sleep: the laterodorsal tegmental nucleus (LDT), dorsal raphe nucleus (DRN), and locus ceruleus (LC). After periods of dark exposure that triggered enrichment of REM sleep, the number of Fos-positive cells in the extended VLPO was highly correlated with REM but not NREM sleep. In contrast, the number of Fos-positive cells in the VLPO cluster was correlated with NREM but not REM sleep. Sixty percent of sleep-active cells in the extended VLPO and 90% of sleep-active cells in the VLPO cluster in dark-treated animals contained galanin mRNA. Retrograde tracing from the LDT, DRN, and LC demonstrated more labeled cells in the extended VLPO than the VLPO cluster, and 50% of these in the extended VLPO were sleep-active. Anterograde tracing showed that projections from the extended VLPO and VLPO cluster targeted the cell bodies and dendrites of DRN serotoninergic neurons and LC noradrenergic neurons but were not apposed to cholinergic neurons in the LDT. The connections and physiological activity of the extended VLPO suggest a specialized role in the regulation of REM sleep.